Anticancer agent comprising anti-pd-1 antibody or anti-pd-l1 antibody

ABSTRACT

Provided is an anticancer agent which comprises an anti-PD-1 antibody or an anti-PD-L1 antibody as an active ingredient, functioning to reverse the unresponsiveness of iNKT cells in which anergy has been induced by administration with an iNKT cell ligand. The anti-PD-1 or anti-PD-L1 antibody blocks the PD-1/PD-L1-mediated signaling pathway not only to prevent the iNKT cell ligand-induced iNKT cell anergy, but also to reverse the unresponsiveness of already anergic iNKT cells to produce cytokines. In addition, the anti-PD1 or anti-PD-L1 antibody ensures the potent anti-tumor activity of iNKT cells as demonstrated by a significant reduction in the number of metastatic nodules in B16F10 melanoma metastasis models in vivo. Collectively, the anticancer agent can be very useful in the treatment of cancer, particularly metastatic cancer.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority from Korean Patent ApplicationNo.10-2008-0097236, filed Oct. 2, 2008, the entire disclosure of whichis incorporated herein by reference in its entirety.

TECHNICAL FIELD

The present invention relates, in general, to an anticancer agent and,more particularly, to an anticancer agent comprising an anti-PD-1antibody or an anti-PD-L1 antibody as an active ingredient, functioningto restore the responsiveness of iNKT cells in which anergy has beeninduced by administration with an iNKT cell ligand.

BACKGROUND OF THE INVENTION

Natural killer T (NKT) cells, co-expressing a T cell receptor and NKcell markers, are essential for several aspects of immunity, such asimmunomodulation and immunuopotentiation, in various immune diseasesincluding autoimmune diseases, infectious diseases, cancer, etc. NKTcells exist at high levels in the thymus, the liver, and the bonemarrow, but at low levels in the spleen, lymph nodes and blood.

Unlike conventional T cells that recognize small peptide antigenspresented by major histocompatibility complex MHC class 1 or MHC class2, NKT cells recognize glycolipid antigens presented by CD1d, a MHCclass 1-like molecule. A major subset of NKT cells, called type 1 NKTcells or invariant natural T (iNKT) cells, express an invariant naturalT cell receptor (TCR) composed of Vα14-Jα18 chains in mice (Vα24-Jα18 inhumans). Upon TCR stimulation with a ligand, such asα-galactosylceramide (α-GC), iNKT cells rapidly produce a wide range ofcytokines including IL-4, IFN-γ, L-12, and GM-CSF. This rapid and potentresponse to a ligand enables iNKT cells to enhance or regulate theactivity of various immune cells in innate and acquired immunity. Foundin diverse diseases and promoting tumor rejection or regulatingautoimmune disorders, these immunomodulatory roles of iNKT cells arestudied for use in immunotherapy treatments for cancer and autoimmunediseases.

However, iNKT cells tend to greatly decrease in responsiveness followingrepeated stimulation after a first stimulation with their ligands viathe T cell receptor. For instance, iNKT cells that have been stimulatedin vivo with a-GC have reduced proliferation and cytokine productionupon secondary stimulation with the same ligand. This iNKT cell anergyis a major obstacle in immunotherapeutic trials targeting iNKT cells.

Conventional T cells are known to become anergic when they receive a TCRsignal with insufficient co-stimulatory signals. Co-stimulatorymolecules such as CD28, CD40L and ICOS are known to be involved in thedevelopment and activation of iNKT cells. Recently, it has been reportedthat 4-1BB contributes to promote the activation of iNKT cells as aco-stimulatory molecule and can affect iNKT cell-mediated allergic lunginflammation (Kim, D. H., W. S. Chang, Y. S. Lee, K. A. Lee, Y. K. Kim,B. S. Kwon, and C. Y. Kang. 2008. 4-1BB engagement co-stimulates NKTcell activation and exacerbates NKT cell ligand-induced airwayhyperresponsiveness and inflammation. J Immunol 180:2062-2068.). On theother hand, it has recently been suggested that coinhibitory molecules,such as PD-1, B7H3, and B7H4, may actively anergize or tolerize T cellsby delivering inhibitory signals into TCR-stimulated T cells. In alymphocytic choriomeningitis virus (LCMV) infected model, CD8 T cellsare tolerized by LCMV epitope-presenting dendritic cells. However, theblockade of the PD-1 signal can reverse the anergic phenotype of CD8 Tcells. It has also been reported that the inhibition of PD-1/PD-L1restores the function of exhausted CD8 T cells in a chronic infectionmodel.

Programmed death-1 (PD-1) is a 55 KDa type 1 transmebrane protein of theimmunoglobulin superfamily, and is known as a co-inhibitory molecule onT cells. That is, PD-1 is a member of the co-inhibitory molecules of theCD28 family (e.g., CD28, CTLA4, ICOS and BTLA) expressed on activated Bcells, T cells and bone marrow cells. Two ligands for PD-1, PD-L1 andPD-L2, have been identified thus far. The interaction of PD-1 with thePD ligands can transduce inhibitory or co-stimulatory signals into the Tcells. In conventional T cells, PD-1 is not expressed on naive T cells,but is inducibly expressed after T cell activation. As for PD-L1, it isexpressed to some degree on naive T cells and its level is increased onactivated T cells. PD-L1 is found at high levels in various humancancers and interacts with PD-1 to transduce inhibitory orco-stimulatory signals from entering into the T cells. For example, theinteraction between PD-1 and PD-L1 induces a decrease in the level oftumor invasive lymphocytes and in T cell receptor-mediated proliferationand causes the immune evasion of tumor cells. In PD-1-deficient animals,PD-1 develops various autoimmune phenotypes, such as autoimmune cardiacinfarction and lupus-like syndromes with arthritis and nephritis, andplays an important role in the development of autoimmuneencephalomyelitis, systemic lupus erythematosus, graft-versus-hostdisease (GVHD), type 1 diabetes and rheumatic arthritis. AgedPD-1-deficient mice develop autoimmune diseases, indicating that PD-1plays a critical role in the regulation of autoimmunity and immunetolerance. In particular, PD-1 signals are essential for inducing T cellexhaustion during chronic infection.

With the ability thereof to stimulate the T cell receptor to rapidlyproduce various cytokines of iNKT cells, the NKT cell ligand α-GC hasconventionally been used as an anticancer agent. However, since repeatedstimulation of iNKT cells with α-GC induces anergy leading to a greatdecrease in responsiveness, it cannot achieve effective anticancereffects. Therefore, there is a pressing need for an anticancer agentthat can restore the responsiveness of iNKT cells even in the state ofanergy caused by stimulation with iNKT cell ligands.

Leading to the present invention, intensive and thorough research intoan anticancer agent taking advantage of the responsiveness of iNKTcells, conducted by the present inventors, resulted in the finding thatPD-1 expressed on iNKT cells is upregulated after stimulation and thatblocking of the PD-1/PD-L1 signaling pathway by an anti-PD-1 oranti-PD-L1 antibody allows iNKT cells under an iNKT cell ligand-inducedanergy condition to recover their responsiveness, such as the productionof cytokines. Also, the anti-PD1 or anti-PD-L1 antibody was found toinduce potent anti-tumor activity of iNKT cells as demonstrated by asignificant reduction in the number of metastatic nodules in B16F10melanoma metastasis models in vivo.

SUMMARY OF THE INVENTION

It is therefore an object of the present invention to provide ananticancer agent which is based on the activity of iNKT cells, featuringthe recovery of iNKT cell responsiveness.

It is another object of the present invention to provide a method forthe treatment of cancer using the same.

BRIEF DESCRIPTION OF THE DRAWINGS

The above and other objects, features and advantages of the presentinvention will be more clearly understood from the following detaileddescription taken in conjunction with the accompanying drawings, inwhich:

FIG. 1 is of flow histograms showing the expression levels of PD-1,PD-L1 and PD-L2 on iNKT cells of the splenocytes isolated fromα-GC-treated C57BL/6 mice at indicated time points (0 hr, 6 hrs, 72 hrs,7 days, 1 month and 2 months) after treatment.

FIG. 2 is of histograms showing IFN-γ and IL-4 levels in thesupernatants obtained after incubating splenocytes isolated from naïveC57BL/6 mice with α-GC in the presence of control rat IgG, anti-PD-1monoclonal antibody, anti-PD-L1 monoclonal antibody or anti-PD-L2monoclonal antibody as analyzed by ELISA (in vitro).

FIG. 3 is of plots showing IFN-γ and IL-4 levels in the sera obtainedfrom the C57BL/6 mice, which were intraperitoneally injected withcontrol rat IgG, anti-PD-1 monoclonal antibody, anti-PD-L1 monoclonalantibody or anti-PD-L2 monoclonal antibody 24 hrs before α-GC treatment,at 0, 2, 6, 12, 24, 48 and 72 hrs after the α-GC treatment as analyzedby ELISA (in vivo).

FIG. 4 shows FACS results of IFN-γ⁺ or IL-₄ ⁺ iNKT cells on whichintracellular cytokine staining was performed to examine if theincreased production of IFN-γ and IL-4 in the sera of FIG. 3 comes fromiNKT cells.

FIG. 5 is of histograms showing IFN-γ and IL-4 levels in thesupernatants obtained after the splenocytes of C57BL/6 mice, in whichiNKT cell anergy was induced by α-GC treatment 7 days and one monthbefore splenocyte isolation therefrom, were incubated with α-GC in thepresence of control rat IgG, anti-PD-1 monoclonal antibody, anti-PD-L1monoclonal antibody or anti-PD-L2 monoclonal antibody, as analyzed byELISA (in vitro).

FIG. 6 is of histograms showing IFN-γ and L-4 levels in sera fromC57BL/6 mice, as analyzed by ELISA. The mice were intraperitoneallyinjected with control rat IgG, anti-PD-1 monoclonal antibody, anti-PD-L1monoclonal antibody or anti-PD-L2 monoclonal antibody, followed bydouble injection with α-GC 24 hrs and 14 days later. The sera wereobtained 2 and 12 hrs after the secondary injection of α-GC (in vivo).

FIG. 7 shows FACS results of IFN-γ⁺ or IL-4⁺ iNKT cells afterintracellular cytokine staining was performed on the splenocytesisolated 2 hrs after the secondary injection (in vivo).

FIG. 8 shows CD69 expression levels on iNKT and NK cells as measured byflow cytometry using the splenocytes isolated 12 hrs after the secondaryinjection (in vivo).

FIG. 9 is a histogram showing weights of normal and metastatic lungs ofC57BL/6 mice. The mice were intraperitoneally injected with control ratIgG, anti-PD-1 monoclonal antibody or anti-PD-L1 antibody 24 hrs beforeintravenous injection with skin tumor cells. On Days 0, 4 and 8, themice were co-administered with α-GC and the control rat IgG, theanti-PD-1 antibody or the anti-PD-L1 antibody. The lungs were excised 14days after the co-administration.

FIG. 10 shows effects of the blockage of PD-1/PD-L1 interaction on theantitumor activity of iNKT cells in terms of the number of metastaticpulmonary nodules (A) and optical microscopic views (B). C57BL/6 micewere intraperitoneally injected with control rat IgG, anti-PD-1 antibodyor anti-PD-L1 antibody 24 hrs before α-GC treatment. 7 days later, themice were intravenously injected with skin tumor cells and re-injectedwith α-GC on Days 0, 4 and 8. 14 days later, the lungs were excised.

DETAILED DESCRIPTION OF THE INVENTION

In accordance with an aspect thereof, the present invention provides ananticancer agent, comprising an iNKT cell ligand as a first anticancerfactor and an anti-PD-1 antibody or anti-PD-L1 antibody as a secondanticancer factor, having a function of reversing unresponsiveness ofiNKT cells in which anergy is induced by administration with the iNKTcell ligand.

In accordance with another aspect thereof, the present inventionprovides a method for reversing the unresponsiveness of iNKT cells withanergy induced therein by iNKT cell ligand treatment, comprisingtreating the anergic iNKT cells with an anti-PD-1 antibody or ananti-PD-L1 antibody.

The iNKT cell ligand may be selected from a group consisting ofalpha-galactosyl ceramide, alpha-glucuronosyl ceramide,phosphatidylinositoltetramannoside, isoglobotrihexosylceramide,ganglioside GD3, phsphatidylcholine, phosphatidylethanolamine,phosphatidylinositol, sulfatide, beta-galactosylceramide,lipophosphoglycan, glycoinositol phospholipid, alpha-galactosylceramideanalogs including beta-anomer galactoceramide and alpha-anomergalactosylceramide, and bacterial lipid antigens.

The anti-PD-1 antibody or the anti-PD-L1 antibody may be a monoclonalantibody or a polyclonal antibody.

The anti-PD-1 antibody or anti-PD-L1 antibody according to the presentinvention can block the signaling of PD-1 or PD-L1 to prevent the iNKTcell ligand-induced anergy of iNKT cells and can provide cytokinesecretion ability for even anergic iNKT cells to restore theirresponsiveness. In addition, the anti-PD-1 antibody or anti-PD-L1antibody of the present invention inhibits the anergy induction of iNKTcells to significantly decrease the number of pulmonary nodules in alung metastasis model of B16F10 melanoma, thus effectively elicitinganticancer immune responses of iNKT cells and showing anti-tumor effectsof iNKT cells against cancer metastasis. Therefore, an anticancer agentcomprising the anti-PD-1 antibody or anti-PD-L 1 antibody of the presentinvention can be very useful in the treatment of cancer, particularly,metastatic cancer.

The cancer to which the anticancer agent according to the presentinvention is therapeutically applicable may be gynecologic tumor,endocrine gland cancer, CNS (central nervous system) tumor or uretercancer. Concrete examples of the cancer include lung cancer, stomachcancer, liver cancer, bone cancer, pancreatic cancer, skin cancer, headand neck cancer, melanoma, uterine cancer, ovarian cancer, rectalcancer, colorectal cancer, colon cancer, breast cancer, uterine sarcoma,Fallopian tube carcinoma, endometrial carcinoma, uterine cervicalcarcinoma, vaginal carcinoma, vulva carcinoma, esophageal cancer, larynxcancer, small intestine cancer, thyroid cancer, parathyroid cancer, softtissue sarcoma, uterine cancer, penis cancer, prostate cancer, chronicor acute leukemia, pediatric solid tumor, differentiated lymphoma,bladder cancer, kidney cancer, renal cell carcinoma, renal pelviccarcinoma, primary CNS lymphoma, spinal cord tumor, brainstem glioma,and pituitary adenoma.

The anticancer agent of the present invention may be formulated into apharmaceutical composition with at least one conventional anticanceringredient.

In addition to the active ingredients, the pharmaceutical compositionmay further comprise a pharmaceutically acceptable carrier. Examples ofthe pharmaceutically acceptable carrier include saline, sterile water,Ringer's solution, buffered saline, dextrose solution, maltodextrinsolution, glycerol, ethanol, etc. Optionally, conventional additives,such as antioxidants, buffers, bacteriostatic agents, etc., may be addedto the composition. For the preparation of dosage forms includinginjections, such as aqueous solutions, suspensions and emulsions, pills,capsules, granules and tablets, the active ingredients may be admixedwith a diluent, a dispersant, a surfactant, a binder and/or a lubricant.Reference may be made to literature (Remington's Pharmaceutical Science(recent edition), Mack Publishing Company, Easton Pa.) upon theformulation of the pharmaceutical composition into suitable dosageforms.

The composition of the present invention may be administered via oralroutes or parenteral routes (e.g., intravenous, subcutaneous,intraperitoneal, topical, etc.). The effective dosage of the anticanceragent in accordance with the present invention depends on variousfactors, including the patient's weight, age, gender, state of health,diet, the time of administration, route of administration, excretionrate, severity of diseases, etc. In general, it may be administered in asingle dose, and preferably in multiple doses per day at a daily doseranging from 0.01 to 1000 mg/day, and preferably from 0.1 to 100 mg/kgof the anti-PD-1 antibody or anti-PD-L1 antibody.

For the effective prophylaxis and treatment of cancer, the compositionaccording to the present invention may be used alone or in combinationwith surgical operation, hormonal therapy, chemotherapy, and/orbiological response controllers.

A better understanding of the present invention may be obtained throughthe following examples which are set forth to illustrate, but are not tobe construed as limiting the present invention.

EXAMPLES Example 1 Expression of PD-1 and PD Ligands in iNKT Cells

1. Experimental Animals

Six- to eight-week-old female C56BL/6 mice were purchased from OrientBio. All mice were bred and maintained in specific pathogen-freeconditions.

2. Reagents and Antibodies

α-GC was dissolved in PBS containing 0.5% Tween 20. Hybridoma clonesproducing antibodies to mouse PD-1 (RMP1-14, rat IgG2a), PD-L1 (MIH-5,rat IgG2a), and PD-L2 (Ty25, rat IgG2a) were produced according tomethods well known in the art [Yamazaki, T., H. Akiba, H. Iwai, H.Matsuda, M. Aoki, Y. Tanno, T. Shin, H. Tsuchiya, D. M. Pardoll, K.Okumura, M. Azuma, and H. Yagita. 2002. Expression of programmed death 1ligands by murine T cells and APC. J Immunol 169:5538-5545.; Tsushima,F., H. Iwai, N. Otsuki, M. Abe, S. Hirose, T. Yamazaki, H. Akiba, H.Yagita, Y. Takahashi, K. Omura, K. Okumura, and M. Azuma. 2003.Preferential contribution of B7-H1 to programmed death-1-mediatedregulation of hapten-specific allergic inflammatory responses. Eur JImmunol 33:2773-2782.; Yamazaki, T., H. Akiba, A. Koyanagi, M. Azuma, H.Yagita, and K. Okumura. 2005. Blockade of B7-H1 on macrophagessuppresses CD4+ T cell proliferation by augmenting IFN-gamma-inducednitric oxide production. J Immunol 175:1586-1592.]. All clones werecultured in RPMI 1640 (Gibco) with 10% FBS (Gibco) and 1%penicillin/streptomycin (BioWhittaker). All antibodies were preparedfrom the ascites of nude mice using caprylic acid purification. Controlrat IgG was also prepared by caprylic acid purification from sera ofnaïve rats.

3. Expression of PD-1 and PD Ligands on iNKT Cells After α-GCAdministration

Splenocytes were isolated from C57BL/6 mice administered with 2 μg ofα-GC at different time points (0 hr, 6 hrs, 72 hrs, 7 days, one monthand two months) after the administration. The cells were stained with aPE-conjugated anti-PD-1 monoclonal antibody, a PE-conjugated anti-PD-L1monoclonal antibody, a PE-conjugated anti-PD-L2 monoclonal antibody anda PE-conjugated isotype control monoclonal antibody, respectively. iNKTcells were gated on B220⁻ TCR-β^(int)α-GC/CD1d:Ig⁺ population. PD-1,PD-L1 and PD-L2 expression (open histograms) was analyzed by FACS. Theresults are given in FIG. 1. Shaded histograms indicate staining withisotype control niAbs.

As depicted in FIG. 1, PD-1 was constitutively expressed on iNKT cellsat a low level and its expression was upregulated after α-GCstimulation, followed by the persistence of the upregulated level fortwo months. On the other hand, PD-L1 expression was temporarilyincreased on α-GC-stimulated iNKT cells but declined toward naïve statelevels within 72 hrs. As for PD-L2, its expression was not found on iNKTcells regardless of their activation status.

Example 2 Effects of PD-1, PD-L1 and PD-L2 on iNKT Cell Activation

The following in vitro and in vivo experiments were conducted to examinethe effects of PD-1, PD-L1 and PD-L2 on iNKT cell activation.

1. iNKT Cell Activation In Vitro

After being prepared from naëve C57BL/6 mice, 5×10⁵ spelenocytes wereincubated for 3 days with 100 ng/ml of α-GC in the presence of 50 μg/mlof the control rat IgG, the anti-PD-1 mAb, the anti-PD-L1 mAb, or theanti-PD-L2 mAb. Then, the supernatants were obtained and assayed forIFN-γ and IL-4 levels by ELISA.

The results are given in FIG. 2.

Compared with control IgG treatment, as is apparent from FIG. 2,anti-PD-1 mAb significantly increased the production of IFN-γ and, to alesser extent, IL-4. Anti-PD-L1 mAb also induced greater production ofIFN-γ, but anti-PD-L2 mAb did not.

2. iNKT Cell Activation in Vivo

C57BL/6 mice was injected with 200 μg of the control rat IgG, theanti-PD-1 mAb, the anti-PD-L1 mAb or the anti-PD-L2 mAb 24 hrs beforetreatment with 2 μg of α-GC. Sera were obtained at 0, 2, 6, 12, 24, 48and 72 hrs after the treatment, followed by ELISA analysis for IFN-γ andIL-4 levels.

In order to determine whether the increased production of IFN-γ wasdistinctively attributed to iNKT cells, intracellular cytokine stainingwas followed by flow cytometry analysis. In this regard, splenocyteswere isolated 2 hrs after α-GC treatment and 5×10⁶ cells were incubatedwith Golgi plug for 2 hrs to accumulate cytokines. Intracellularcytokine staining was performed using BD Cytofix/Cytoperm Plus withGolgiplug kit according to the manufacturer's protocol (BD Biosciences).iNKT cells were gated on B220⁻TCR-β^(int) α-GC/CD1d:Ig⁺ population andIFN-γ⁺ or IL-4⁺ iNKT cells were analyzed by flow cytometry.

IFN-γ and IL-4 levels in blood are depicted in FIG. 3 and flowhistograms of IFN-γ⁺ and IL-4⁺ iNKT cells are given in FIG. 4.

As shown in FIG. 3, the blood IFN-γ and IL-4 levels were observed topeak, respectively, within 12 hrs and 2 hrs after α-GC treatment,indicating iNKT cell activation. IFN-γ levels in the sera of anti-PD-1or anti-PD-L1 mAb-treated mice were significantly enhanced compared withlevels in the group treated with control IgG. Slightly different L-4production was observed after treatment with the blocking Abs, but withno significance.

As shown in FIG. 4, iNKT cells producing IFN-γ increased in numberbecause they blocked PD-1/PD-L1 interaction with the anti-PD-1 mAb oranti-PD-L1 mAb in comparison with control cells. On the other hand, nosignificant differences were found between populations of IL-4⁺ iNKTcells and control cells after mAb treatment. These data indicate thatthe blockage of PD-1/PD-L1 interaction allows the delivery of theco-inhibitory signal during iNKT activation, resulting in increasedIFN-γ secretion from iNKT cells.

Example 3 Effects of Blockage of PD-1/PD-L1 Interaction onResponsiveness of Anergic iNKT Cells

The following in vitro and in vivo experiments were performed in orderto examine whether the blockage of PD-1/PD-L 1 interaction reverses iNKTcell anergy.

1. Recovery of Responsiveness of Anergic iNKT Cells in Vitro

The α-GC-induced unresponsiveness of iNKT cells was detected as early as3 days after primary stimulation and observed to persist until 7˜30 daysafter α-GC stimulation. Thus, C57BL/6 mice were injected with 2 μg ofα-GC to induce iNKT cell anergy. One week and one month later,splenocytes were isolated from the mice and 5×10⁵ cells were incubatedfor 3 days with 100 ng/ml of α-GC in the presence of 50 μg/ml of thecontrol rat IgG, the anti-PD-1 mAb, the anti-PD-L1 mAb, or theanti-PD-L2 mAb. Also, splenocytes isolated from the mice were incubatedfor 3 days with 10 ng/ml of α-GC without mAb, and these were representedby ‘activation’. The supernatants were then assayed for IFN-γ and IL-4levels by ELISA [*:p<0.05 and **:p<0.01 (vs. control rat IgG)].

The results are depicted in FIG. 5.

As seen in the graphs of FIG. 5, when restimulated with α-GC in thepresence of the control IgG, the splenocytes of the mice in which iNKTcell anergy was induced by pre-treatment with α-GC 7 days before therestimulation were found to greatly decrease in the production of IFN-γand IL-4. In contrast, the restimulation of iNKT cell anergy-inducedsplenocytes with α-GC in the presence of the anti-PD-1 antibody oranti-PD-L1 antibody resulted in a great increase in the production ofIFN-γ and L-4 (A). This upregulation was observed to persist for one totwo months after α-GC treatment (B). Therefore, the blockage ofPD-1/PD-L1 interaction during restimulation with α-GC reverses theestablished anergic phenotype of iNKT cells.

2. Recovery of Responsiveness of Anergic iNKT Cells in Vivo

200 μg of the control rat IgG, the anti-PD-1 antibody or the anti-PD-L1antibody was intraperitoneally injected into C57BL/6 mice 24 hrs beforetreatment with 2 μg of α-GC. 14 days later, 2 μg of α-GC was injectedagain, followed by the preparation of sera 2 and 12 hrs after there-injection for ELISA assay of IL-4 and IFN-γ levels, respectively.

Splenocytes (5×10⁶ cells), prepared two and twelve hours after thesecond injection of α-GC, were incubated with Golgi plug for 2 hrs toaccumulate cytokines. Intracellular cytokine staining was performed onthe splenocytes prepared 2 hrs after the secondary GC treatment, usingBD Cytofix/Cytoperm Plus with Golgiplug kit according to themanufacturer's protocol (BD Biosciences). The splenocytes prepared 12hrs later were used in an assay for CD69 expression in iNKT and NKcells. iNKT cells were gated on B220⁻TCR-β^(int)α-GC/CD1d:1g⁺ populationand NK cells were gated on B220⁻ TCR-β⁻NK1.1^(high) population. IFN-γ⁺or IL-4⁺ iNKT cells were analyzed by flow cytometry. Also, CD69expression on iNKT and NK cells was analyzed by flow cytometry.

IFN-γ and L-4 levels in blood are graphed in FIG. 6 and FACS results ofIFN-γ⁺ or L-4⁺ iNKT cells and flow histograms of CD69 expression on iNKTand NK cells are given in FIGS. 7 and 8, respectively.

Like mice treated with control rat IgG, as seen in FIG. 6, micepretreated with α-GC produced significantly low IFN-γ and L-4 uponsecondary α-GC injection. In contrast, mice treated with the anti-PD-1antibody during the primary α-GC injection produced remarkably higherIFN-γ and L-4 upon the secondary α-GC injection than did the controlIgG-treated group.

As seen in FIG. 7, the increased cytokine production was attributed toiNKT cells.

It was also found that CD69 expression upon secondary α-GC injection wasincreased on iNKT and NK cells in a similar manner by the treatment withanti-PD-1 mAbs, as shown in FIG. 8. Thus, PD-1/PD-L1 interaction isessential for the induction of iNKT cell anergy in vivo.

Example 4 Effects of Anti-PD-1 mAb or Anti-PD-L1 mAb on Anti-TumorActivity of Activated iNKT Cells

The following experiments were performed to investigate the effects ofthe anti-PD-1 antibody or the anti-PD-L1 antibody on the anticanceractivity of activated iNKT cells in a B16F10 melanoma metastasis model.

1. Weight of Lung

Skin tumor cells (B16F10, ATCC) were cultured in DMEM media supplementedwith 10% FBS and 1% penicillin/streptomycin. C56BL/6 mice wereintraperitoneally injected with 200 g of the control rat IgG, theanti-PD-1 mAb orthe anti-PD-L1 mAb 24 hrs before i.v. inoculation with2×10⁵ tumor cells. On Days zero, 4 and 8, the mice were treated with 500ng of α-GC plus 200 μg of the control rat IgG, the anti-PD-1 mAb or theanti-PD-L1 mAb. On Day 14, the lungs were weighed and weight differencesbetween metastatic and normal lungs are graphed in FIG. 9.

As depicted in FIG. 9, the B16F10 metastatic lungs from the grouptreated with α-GC plus the anti-PD-1 mAb were significantly reduced inweight compared with those from the group treated with α-GC plus thecontrol rat IgG.

2. Number of Pulmonary Nodules

Anergy-induced iNKT cells were assayed for the recovery of anti-tumoractivity by the antibodies of the present invention. In this regard,naive C57BL/6 mice were intraperitoneally injected with 200 μg of thecontrol rat IgG, the anti-PD-1 antibody or the anti-PD-L1 antibody 24hrs after which injection with 2 μg of α-GC induced iNKT cell anergy. 7days later, 5×10⁵ B16F10 tumor cells were i.v. injected. On Day 0, 4 and8, the mice were treated with 500 ng of α-GC to induce anti-tumoractivity of iNKT cells. 14 days later, the lungs were excised and thenodules formed by cancer metastasis were counted (A). The isolated lungswere observed under an optical microscope (B). The results are given inFIG. 10.

As seen in FIG. 10, when induced by treatment with the control IgG andα-GC, iNKT cell anergy was unchanged with time to show a metastasisresult comparable to that of the normal metastasis control group,indicating that α-GC treatment during tumor inoculation did not suppresstumor growth. In contrast, when the anergy induction of α-GC wasprevented by treatment with the anti-PD-1 antibody or the anti-PD-L1,the numbers of tumor nodules in the B16F10 melanoma metastasis modelwere remarkably reduced compared with those upon treatment with thecontrol IgG, demonstrating the superior anti-tumor activity of theantibodies of the present invention. Taken together, the data obtainedabove indicates that the blockage of PD-1/PD-L1 interaction by treatmentwith the anti-PD-1 antibody or the anti-PD-L1 antibody during theinduction phase of iNKT cell anergy leads to the persistence of theanti-tumor effects of iNKT cells.

Formulation examples are given to illustrate dosage preparationscontaining the anticancer agent of the present invention.

Formulation Example 1 Preparation of Powder

Anti-PD-1 or Anti-PD-L1 Antibody 0.1 g Lactose 1.5 g Talc 0.5 g

These ingredients were mixed and loaded into an airtight sac to give apowder.

Formulation Example 2 Preparation of Tablet

Anti-PD-1 or Anti-PD-L1 Antibody 0.1 g Lactose 7.9 g Crystallinecellulose 1.5 g Magnesium stearate 0.5 g

These ingredients were mixed and directly compressed into a tablet.

Formulation Example 3 Preparation of Capsule

Anti-PD-1 or Anti-PD-L1 Antibody 0.1 g Corn starch   5 gCarboxycellulose 4.9 g

These ingredients were admixed together and the admixture was loadedinto a conventional capsule using a suitable device.

Formulation Example 4 Preparation of Injection

Anti-PD-1 or Anti-PD-L1 Antibody 0.1 g Sterile water for injectionproper quantity pH Adjuster proper quantity

Using a conventional method, these ingredients were put into an ampule(2 ml) to give an injection.

Formulation Example 5 Preparation of Liquid Medicine

Anti-PD-1 or Anti-PD-L1 Antibody 0.1 g Isomerized sugar  10 g Mannitol  5 g Purified water proper quantity

Each ingredient was dissolved in purified water and flavored with lemonbefore admixing together. Purified water was added to the admixture toform a final volume of 100 ml which was then loaded into a brown vialand sterilized.

As described hitherto, the anti-PD-1 or anti-PD-L1 antibody according tothe present invention blocks the PD-1/PD-L1-mediated signaling pathwaynot only to prevent the iNKT cell ligand-induced iNKT cell anergy, butalso to reverse the unresponsiveness of already anergic iNKT cells toproduce cytokines. In addition, the anti-PD1 or anti-PD-L1 antibodyensures the potent anti-tumor activity of iNKT cells as demonstrated bya significant reduction in the number of metastatic nodules in B16F10melanoma metastasis models in vivo. Collectively, the anticancer agentcomprising an anti-PD-1 or anti-PD-L1 antibody in accordance with thepresent invention can be very useful in the treatment of cancer,particularly metastatic cancer.

Although the preferred embodiments of the present invention have beendisclosed for illustrative purposes, those skilled in the art willappreciate that various modifications, additions and substitutions arepossible, without departing from the scope and spirit of the inventionas disclosed in the accompanying claims.

1. An anticancer agent, comprising an iNKT cell ligand as a firstanticancer factor and an anti-PD-1 antibody or anti-PD-L1 antibody as asecond anticancer factor, having a function of reversingunresponsiveness of iNKT cells in which anergy is induced byadministration with the iNKT cell ligand.
 2. The anticancer agent asdefined in claim 1, wherein the iNKT cell ligand is selected from agroup consisting of alpha-galactosyl ceramide, alpha-glucuronosylceramide, phosphatidylinositoltetramannoside,isoglobotrihexosylceramide, ganglioside GD3, phsphatidylcholine,phosphatidylethanolamine, phosphatidylinositol, sulfatide,beta-galactosylceramide, lipophosphoglycan, glycoinositol phospholipid,alpha-galactosylceramide analogs including beta-anomer galactoceramideand alpha-anomer galactosylceramide, and bacterial lipid antigens. 3.The anticancer agent as defined in claim 1, wherein the anti-PD-1antibody or the anti-PD-L1 antibody is a monoclonal antibody or apolyclonal antibody.
 4. The anticancer agent as defined in claim 1,wherein the cancer is selected from a group consisting of lung cancer,stomach cancer, liver cancer, bone cancer, pancreatic cancer, skincancer, head and neck cancer, melanoma, uterine cancer, ovarian cancer,rectal cancer, colorectal cancer, colon cancer, breast cancer, uterinesarcoma, Fallopian tube carcinoma, endometrial carcinoma, uterinecervical carcinoma, vaginal carcinoma, vulva carcinoma, esophagealcancer, larynx cancer, small intestine cancer, thyroid cancer,parathyroid cancer, soft tissue sarcoma, uterine cancer, penis cancer,prostate cancer, chronic or acute leukemia, pediatric solid tumor,differentiated lymphoma, bladder cancer, kidney cancer, renal cellcarcinoma, renal pelvic carcinoma, primary CNS lymphoma, spinal cordtumor, brainstem glioma, and pituitary adenoma.
 5. A method forreversing the unresponsiveness of iNKT cells with anergy induced thereinby iNKT cell ligand treatment, comprising treating the anergic iNKTcells with an anti-PD-1 antibody or an anti-PD-L1 antibody.
 6. Themethod as defined in claim 5, wherein the iNKT cell ligand is selectedfrom a group consisting of alpha-galactosyl ceramide, alpha-glucuronosylceramide, phosphatidylinositoltetramannoside,isoglobotrihexosylceramide, ganglioside GD3, phsphatidylcholine,phosphatidylethanolamine, phosphatidylinositol, sulfatide,beta-galactosylceramide, lipophosphoglycan, glycoinositol phospholipid,alpha-galactosylceramide analogs including beta-anomer galactoceramideand alpha-anomer galactosylceramide, and bacterial lipid antigens. 7.The method as defined in claim 5, wherein the anti-PD-1 antibody or theanti-PD-L1 antibody is a monoclonal antibody or a polyclonal antibody.